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Abstract

Background

Outbreaks of food poisoning associated with drinking un-pasteurised apple juice contaminated
with enterohaemorrhagic Escherichia coli O157:H7 are a cause of serious illness and occasionally death. Whilst a well-established
heat process (pasteurisation) will readily eliminate the pathogen, some consumers
are demanding more fresh-like foods that have not been subjected to processing methods
that are perceived as severe and may lead to loss of flavour and vitamins. Therefore,
alternative methods are being investigated to replace pasteurisation and improve the
safety of minimally-processed juices. The addition of natural antimicrobial substances
such as the phenolic substances carvacrol and p-cymene (derived from the essential oils of herbs and spices) provides a potential
new route to assure safety and extend the shelf-life of raw fruit juices.

The aim of this study was to evaluate the addition of very low concentrations (0.25–1.25
mM) of carvacrol and p-cymene both individually and in combination as a novel means of controlling Escherichia coli O157:H7 in un-pasteurised apple juice.

Results

When inoculated at a level of 4 log CFU/ml into un-pasteurised apple juice (pH 3.20
± 0.06), Escherichia coli O157:H7 survived for up to 3 and 19 days at 25° and 4°C, respectively. Treatment of
the juice with 1.25 mM carvacrol or p-cymene reduced the numbers of E. coli O157:H7 to undetectable levels within 1–2 days at both storage temperatures. The effective
concentrations of carvacrol could be reduced even further by combining it at 0.5 mM
with cymene at 0.25 mM. The phenolic compounds were biocidal against both spoilage
yeasts and E. coli O157:H7 thereby increasing the shelf-life and improving the safety of un-pasteurised
apple juice, particularly when stored at chill temperatures.

Conclusion

The results showed that the natural antimicrobial compounds carvacrol and p-cymene
could potentially be used to extend the shelf life and improve the safety margins
in un-pasteurised chilled fruit juices.

Background

Conventional fruit juice processing involves a heating step to inactivate the vegetative
forms of pathogenic and spoilage microorganisms. Any remaining bacterial spores are
generally unable to germinate due to the acidic nature of the juices [1]. This ensures acceptable safety margins and extends the shelf-life of the juice.
However, heat treatment causes vitamin losses and changes in flavour of the juices
and some consumers regard heat-treated, shelf-stable products as low in quality. In
the last 15 years, there have been several outbreaks of food poisoning associated
with drinking un-pasteurised apple juice contaminated with enterohaemorrhagic Escherichia coli O157:H7 and several children have died in the USA [2]. Warning labels are now required in the USA for all fruit juices unless a 5-log pathogen
reduction treatment has been applied to the product [3-5]. Therefore, there is a need for new methods of fruit juice preservation that rely
less heavily on severe heat treatment or the addition of synthetic preservatives.
It has been suggested that many natural antimicrobial compounds from plant, animal
and microbial sources might fulfil this need [6-8].

Carvacrol (2-methyl-5-(1-methylethyl)-phenol) is a major component of the essential
oils of oregano and thyme [9,10]. Generally recognized as a safe food additive, carvacrol is used as a flavouring
agent in baked goods, sweets, beverages and chewing gum [11]. Carvacrol-containing essential oils are biostatic and/or biocidal against many bacteria,
yeast and fungi in laboratory media and consequently have attracted considerable research
attention as potential food preservatives [reviewed in [12]]. Carvacrol has also been shown to inactivate microorganisms in biofilms on stainless
steel surfaces [13,14]. The biocidal mode of action of carvacrol on bacteria is similar to that of other
phenolic compounds and occurs via membrane damage resulting in an increase in membrane permeability to protons and potassium
ions, depletion of the intracellular ATP pool and disruption of the proton-motive
force [15,16].

The biological precursor of carvacrol, p-cymene (1-methyl-4-(1-methylethyl)-benzene), is less antimicrobial than carvacrol
when used alone. p-Cymene lacks a hydroxyl group, which is thought to play an important role in antimicrobial
activity [17]. Synergism between carvacrol and p-cymene against B. cereus in vitro and in rice has been reported [18].

The minimum inhibitory concentrations (MICs) of carvacrol, thymol, cinnamic acid and
other phenolic compounds from herbs and spices against some food-borne bacteria in vitro have been reported at around 1 mM [12,18,19]. However, a much higher concentration is usually needed to achieve the same biocidal
or biostatic effects in foods. For example, the MIC of carvacrol in mushroom soup
inoculated with B. cereus was 50 times higher than that found in laboratory media [20]. Similarly, the concentrations of carvacrol and cinnamic acid required to delay spoilage
of mango, kiwifruit and melon slices was >5 mM [21,22]. A number of intrinsic (fat and protein content, pH, salt, etc.) and extrinsic (temperature,
oxygen availability, etc.) factors are thought to play a role in protecting microorganisms
from the biocidal effects of carvacrol in foods. Furthermore, the potent aromatic
and antioxidant properties of phenolic compounds at these high concentrations have
been reported to lead to undesirable colour, odour and flavour changes in food products
[8,22].

The aim of this study was to evaluate the addition of very low concentrations of carvacrol
and p-cymene both individually and in combination as a novel means of controlling Escherichia coli O157:H7 in un-pasteurised apple juice.

Results

Inactivation/inhibition of microorganisms with 1.25 mM carvacrol and p-cymene used individually

At 25°C, the total viable counts in un-pasteurized, untreated apple juice increased
in the first 4 days to a level of 8 log CFU/ml, where they remained up to Day 7 (Figure
1). Yeast counts reflected the total counts. When inoculated into raw apple juice,
E. coli O157:H7 survived for up to 3 days at 25°C but by Day 4, numbers decreased to below
the detection limit of the plate-counting method (0.5 log CFU/ml). Enrichment methods
allowed the detection of E coli up to 3 days after inoculation at 25°C but by Day 4 (and beyond to Day 20) none were
detected (Table 1).

Figure 1.Survival/growth of microorganisms in apple juice treated with 1.25 mM carvacrol or
p-cymene at 25° and 4°C. Data points represent: control with no additions (●); control inoculated with E. coli O157:H7 (○); juice inoculated with E. coli O157:H7 and treated with 1.25 mM carvacrol (▲); juice inoculated with E. coli O157:H7 and treated with 1.25 mM p-cymene (■). Sampling of control batches at 25°C was discontinued after 7 d due tovisible
spoilage. Dotted line represents the lower detection limit of the plating technique.
The pH of the juice was 3.17 on Day 0 and ranged between 3.30 and 3.44 on the final
day of sampling (Days 7 and 20 at 4° and 25°C, respectively).

Table 1. Presence/Absence of E. coli in apple juice stored at 25°C (A) and 4°C (B). Results (in duplicate) were obtained
by enrichment of samples from the experiment illustrated in Figure 1.

In the presence of 1.25 mM carvacrol, there was an initial reduction (within the first
two days) in total and yeast counts of about 2 log CFU/ml (Figure 1). This was followed by re-growth of total viable numbers to about 6 log CFU/ml within
7 days where they remained steady until Day 20. Yeast numbers continued to fall in
the presence of 1.25 mM carvacrol and by Day 4 reached levels below the detection
limit of the assay (0.5 log CFU/ml). In the presence of 1.25 mM p-cymene, total plate counts increased more slowly than in the untreated controls but
by Day 12, numbers were similar to those in the controls. Yeasts were initially inactivated
by p-cymene and remained at low levels (1–2 log CFU/ml) from Day 1 to 4 but then increased
to about 6–7 log CFU/ml by Day 8 where they remained up to Day 20. Notably, numbers
of E. coli decreased to below the limit of detection of the plate-counting method within less
than one day of exposure to both carvacrol and p-cymene. E. coli were no longer detectable by enrichment methods after 2 days in the presence of carvacrol
and after 3 days in the presence of p-cymene (Table 1).

At 4°C, total viable numbers and yeasts in the untreated juice remained at about 4–5
log CFU/ml for the first 12 days of incubation, followed by very slow growth to about
7 log CFU/ml by Day 20 (Figure 1). In the presence of 1.25 mM carvacrol and p-cymene, there was a gradual decline
in total and yeast numbers with carvacrol affecting a slightly steeper reduction in
total viable numbers than p-cymene. By Day 20, total numbers were about 1 and 4 log CFU/ml in the presence of
1.25 mM carvacrol and p-cymene, respectively. Notably, E. coli O157:H7 survived at a level of 3–4 log CFU/ml for up to 14 days at 4°C, was still
countable on Day 18 (1 log CFU/ml) and was detectable (by enrichment) on Day 19 of
incubation. In contrast, E coli was not countable (above the detection limit of 0.5 log CFU/ml) or detectable (by
enrichment) after 1 day of exposure to either carvacrol or p-cymene (Figure 1). The results for E. coli shown in Figure 1 were obtained using thin TSA overlaid on selective CT-SMAC agar (see Methods section)
in an attempt to resuscitate injured organisms. The results obtained on plain CT-SMAC
agar and on Chromagar (with and without a thin agar layer on top) were very similar
and are consequently not illustrated.

It was noted that the addition of either carvacrol or cymene at 1.25 mM imparted an
intense "spicy" aroma to the treated apple juices.

Since the results above indicated that survival of E. coli O157:H7 was substantially extended at chill temperatures and therefore represented
a greater cause for concern than survival at ambient temperatures, further work was
undertaken at 4°C only. In order to study the effect of combinations of carvacrol
and cymene, both compounds were used at concentrations that had no or very little
inhibitory effect on microorganisms. As shown in Figure 2, addition of 0.5 mM carvacrol or 0.25 mM p-cymene individually to apple juice had a very slight inhibitory effect on total microbial
counts at 4°C. Like in Figure 1, survival of E. coli O157:H7 persisted for two weeks at this temperature (Figure 2 and Table 2). Using the plate-counting technique, it is shown in Figure 2 that survival of E. coli was curtailed by both 0.5 mM carvacrol and 0.25 mM p-cymene to about 5 days. Enrichment
techniques showed that E. coli was detectable up to 7 and 14 days in the presence of 0.5 and 0.25 mM carvacrol and
p-cymene, respectively. However, when the two compounds were added to the juice together,
E. coli could not be counted or detected after 1 day of exposure to the treatment (Figure
2 and Table 2). Plating out on selective agars with or without a thin layer of TSA gave very similar
results to those shown in Figure 2 and so these counts are not illustrated.

Figure 2.Survival of microorganisms in apple juice treated with 0.5 mM carvacrol and/or 0.25
mM p-cymene at 4°C. Data points represent: control with no additions (●); control inoculated with E. coli O157:H7 (○); juice inoculated with E. coli O157:H7 and treated with 0.5 mM carvacrol (▲); juice inoculated with E. coli O157:H7 and treated with 0.25 mM cymene (■); and juice inoculated with E. coli O157:H7 and treated with the combination of 0.5 mM carvacrol plus 0.25 mM cymene (◆).
The dotted line represents the lower detection limit of the plating technique. The
pH of the juice was 3.21 on Day 0 and ranged between 3.16 and 3.32 on the final day
of sampling (Day 20).

Table 2. Presence/Absence of E. coli in apple juice stored at 4°C. Results (in duplicate) were obtained by enrichment of
samples from the experiment illustrated in Figure 2.

It was noted that the addition of 0.5 mM carvacrol and 0.25 mM p-cymene, alone or
in combination, imparted a very slight "spicy" aroma to the treated juices.

Throughout this study, the pH of the juices remained essentially unchanged at around
3.20 ± 0.06, irrespective of the type of treatment or storage temperature.

Discussion

Freshly-pressed apple juice prepared from healthy fruit can be expected to contain
around 3–5 log CFU/ml of viable microorganisms, of which the majority are yeasts [23]. The results in this study were consistent with this expectation, as shown in Figures
1 and 2. The pH of the juices used in this study was at the lower end (3.20 ± 0.06) of the
expected pH range (2.9–4.5) for fresh apple juice [23].

Despite the low pH, E. coli O157:H7 survived in raw apple juice for 3 days at 25°C and nearly 3 weeks (19 d) at
4°C. These findings agree with those reported elsewhere [23-28]. The acidic nature of apple juice does not ensure its safety as E. coli O157:H7 may survive for extended periods of time, especially at chill temperatures.

The antimicrobial properties of carvacrol and similar phenolic compounds from the
essential oils of herbs and spices have been reported previously against individual
microorganisms, tested in vitro [12,20,29-32]. The application of carvacrol in the preservation of some foods such as rice and
fresh-cut fruit has also been reported [18,22]. However, this is the first report of the successful application of relatively low
doses (0.25–1.25 mM) of carvacrol and p-cymene against E. coli O157:H7 in the presence of the mixed microflora of fruit juice. The results demonstrate
that the addition of 1.25 mM carvacrol or p-cymene to apple juice inactivated E. coli O157:H7 within less than 1 day from about 4 log CFU/ml to levels that were undetectable
using conventional microbiological techniques (Figure 1). Furthermore, once inactivated, E. coli remained undetectable for the duration of the trial (20 days; Table 1). In addition, the results obtained using the thin agar layer method for resuscitating
injured organisms were no different from those using selective media alone, suggesting
that the cellular injury caused by the phenolic compounds was irreversible. The substantial
reduction in the number of days (from 19 to 1) that E. coli O157:H7 was able to survive in apple juice when treated with 1.25 mM carvacrol or
p-cymene and stored at chill temperatures represents an opportunity to improve the safety
of un-pasteurised fruit juices.

In addition to their antibacterial activity, carvacrol and p-cymene were also biocidal against the yeast flora naturally present in the apple juice
(Figure 1). However, 1.25 mM p-cymene was not as effective as the same concentration of carvacrol in eliminating
the yeast population at ambient temperature. Those yeasts surviving the treatment
with p-cymene recovered and eventually reached the same viable numbers as in the untreated
control. By contrast, the presence of either phenolic compound at 1.25 mM resulted
in similar, gradual die-off of the yeast population at chill temperatures. The results
confirm the broad antimicrobial spectrum of carvacrol but also suggest that yeasts
may be somewhat less sensitive to phenolic compounds than bacteria. The reduction
in numbers of spoilage yeasts would clearly benefit both the manufacturer and consumer
by extending the shelf-life of the product.

When carvacrol and p-cymene were added to apple juice at 0.5 and 0.25 mM, respectively, E. coli O157:H7 and the yeast population were reduced but to a different extent than was observed
at the higher concentrations of 1.25 mM. The shapes of the inactivation curves for
the total viable numbers (Figure 2) would suggest that there may have been some synergism between carvacrol and p-cymene but the results for E. coli (Figure 2 and Table 2) suggest that the effect may have been additive. In the presence of 0.5 mM carvacrol
and 0.25 mM p-cymene used alone, E. coli was not detectable for 13 and 6 d longer than in the control but in the presence of
both compounds used in combination, the organism was not detectable for 18 d longer.
Therefore, it is not possible to conclude unequivocally whether the effect of adding
the two substances was synergistic or merely additive.

It has been reported previously that carvacrol was more effective in reducing the
viable count of the natural microflora on kiwifruit (pH 3.2–3.6) than on honeydew
melon (pH 5.4–5.5) [22]. At low pH, the hydrophobicity of essential oil components increases, enabling them
to partition more easily into the lipids of the cell membrane of bacteria.

It is known that direct plating on selective media following exposure to physical
or chemical stresses can lead to gross underestimation of viable counts by as much
as 3–4 log CFU ml-1 [33,34]. Although a thin agar layer method [35] and enrichment techniques [36] were used in this study to allow some resuscitation of the inoculated pathogen, it
is possible that the results shown in Figures 1 and 2 represent an underestimate of numbers. Furthermore, it is possible that a larger
sample volume (25 ml instead of 2.5 ml) in the pre-enrichment step would have allowed
the resuscitation and recovery of an even greater number of E. coli from the control juices.

In the present study, ethanol was used as a solvent for the preparation of stock solutions
of carvacrol and cymene. Whilst the final concentrations of ethanol in the juices
were below the tolerance limit previously reported for E. coli [37], it is possible that the low levels of ethanol present (0.95, 1.9 and 4.75% in the
presence of 0.25, 0.5 and 1.25 mM carvacrol/cymene, respectively) may have potentiated
the biocidal action of the phenolic compounds. This possibility would need experimental
confirmation in future work.

Conclusion

When inoculated into un-pasteurised apple juice, E. coli O157:H7 survived for up to 3 and 19 days at 4° and 25°C, respectively. At 1.25 mM
and at both storage temperatures, carvacrol and p-cymene reduced the numbers of E. coli O157:H7 to undetectable levels within 1–2 days. The effective concentrations of carvacrol
could be reduced even further by combining it at 0.5 mM with p-cymene at 0.25 mM. Carvacrol and p-cymene were biocidal against both spoilage yeasts and E. coli O157:H7 thereby increasing the shelf-life and improving the safety of un-pasteurised
apple juice, particularly when stored at chill temperatures.

Methods

Materials

Freshly pressed, unclarified, raw juice from a mixture of Bramley and Cox apples was
obtained directly from a manufacturer in Suffolk, England. The pH of the juice was
3.20 ± 0.06 on arrival at the laboratory. All microbiological media and diluents were
from Oxoid (Basingstoke, UK) and all chemicals from Sigma Chemicals Co. Ltd. (Poole,
Dorset, UK) unless otherwise indicated.

Un-pasteurised apple juice is often referred to as "cider" in the USA. This should
not be confused with UK cider, which is a fermented alcoholic beverage. In this paper,
the term "apple juice" refers strictly to the raw, unprocessed, unfermented juice
of the apple.

Treatment of apple juice with carvacrol and cymene

Containers (200 ml capacity) of apple juice (100 ml) were prepared in duplicate for
each treatment and stored at 4°C and 25°C. Stock solutions (25 mM) of carvacrol and
cymene were prepared in 95% ethanol and added to the apple juice to give final concentrations
of 0.25, 0.5 and/or 1.25 mM. The juice was inoculated with the washed suspension (prepared
as described above) of E. coli O157:H7 to give an approximate count of 104 CFU/ml. Control juices inoculated with E. coli O157:H7 but containing no antimicrobials, as well as absolute controls containing
no antimicrobials or E. coli O157:H7 were also prepared.

Samples (10 ml) were taken periodically for up to 20 days from each container for
microbiological analysis and pH determination. Serial (1:10) dilutions were prepared
in sterile MRD. Total viable numbers were determined by pour-plating (1.0 ml) on PCA.
Yeasts and moulds were enumerated by spread-plating (0.1 ml) on Tryptone Glucose Yeast
Extract Agar (TGYEA) supplemented with chloramphenicol (0.1 mg/ml). PCA and TGYEA
plates were incubated at 30°C for 2–3 days. Single samples were removed from each
duplicate batch of juice periodically during incubation for viable counting and plating
out in triplicate; therefore, mean counts for each time point were calculated from
six replicate determinations.

Pre-enrichment and enrichment steps were used to detect low levels of E. coli O157:H7 in the apple juice following treatment with carvacrol and/or cymene. An aliquot
of 2.5 ml of juice was added to 22.5 ml BPW and incubated at 37°C for 24 h (in the
case of the un-inoculated control, 25 ml apple juice was added to 225 ml BPW). Following
incubation, an aliquot (1 ml) was dispensed into 10 ml EC broth (reduced bile salts
supplemented with novobiocin) and incubated at 37°C for 24 h. A loopful of the EC
broth was streaked onto Sorbitol MacConkey (SMAC) Agar or onto CHROMagar (M-Tech Diagnostic
Ltd., EE-220-Trial; both agars supplemented with cefixime and potassium-tellurite)
and incubated at 37°C for 24 h.

For enumeration of injured cells of E. coli O157:H7, the Thin Agar Layer method (TAL) was used [35]. Spread plates were prepared on selective agars (CT-SMAC agar or CHROMagar supplemented
with cefixime and potassium-tellurite) and on selective agars overlaid with TSA agar.
The number of injured cells was calculated by subtracting the viable count on selective
agar alone from the viable count obtained on the overlaid agar.

Authors' contributions

GK participated in the design of the study and carried out all the experimental work.

SR conceived the study and participated in its design and coordination.

GK and SR drafted the manuscript jointly.

Acknowledgements

The financial support of the European Commission in the form of a Marie Curie Individual
Fellowship awarded to author Kiskó (QLK1-CT-2000-51126) to undertake this work in
author Roller's laboratory in London is gratefully acknowledged.